Toshiharu Nagatsu

Tyrosine hydroxylase (TH), which was discovered at the National Institutes of Health (NIH) in 1964, is a tetrahydrobiopterin (BH4)-requiring monooxygenase that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines (CAs), such as dopamine, noradrenaline, and adrenaline. Since deficiencies of dopamine and noradrenaline in the brain stem, caused by neurodegeneration of dopamine and noradrenaline neurons, are mainly related to non-motor and motor symptoms of Parkinson's disease (PD), we have studied human CA-synthesizing enzymes [TH; BH4-related enzymes, especially GTP-cyclohydrolase I (GCH1); aromatic l-amino acid decarboxylase (AADC); dopamine beta-hydroxylase (DBH); and phenylethanolamine N-methyltransferase (PNMT)] and their genes in relation to PD in postmortem brains from PD patients, patients with CA-related genetic diseases, mice with genetically engineered CA neurons, and animal models of PD. We purified all human CA-synthesizing enzymes, produced their antibodies for immunohistochemistry and immunoassay, and cloned all human genes, especially the human TH gene and the human gene for GCH1, which synthesizes BH4 as a cofactor of TH. This review discusses the historical overview of TH, BH4-, and other CA-related enzymes and their genes in relation to the pathophysiology of PD, the development of drugs, such as l-DOPA, and future prospects for drug and gene therapy for PD, especially the potential of induced pluripotent stem (iPS) cells.

Dopamine (DA) supplementation therapy by L-dopa for Parkinson's disease (PD) was established around 1970. The dose of L-dopa can be reduced by the combined administration of inhibitors of peripheral L-amino acid decarboxylase (AADC), catechol O-methyltransferase (COMT). or monoamine oxidase B (MAO B). DA in the striatum may be produced from exogenously administered L-dopa by various AADC-containing cells. Such us serotonin neurons. The long-term administration of L-dopa in PD patients may produce L-dopa-induced dyskinesia (LID). which may be due to chronic overstimulation of supersensitive DA D1 receptors. L-dopa may be used in combination with various new strategies Such as gene therapy or transplantation in the future. (c) 2008 Elsevier Ltd. All rights reserved.

Several reports on different in vitro and in vivo models have revealed neuroprotective effects afforded by nicotine treatment. Nicotine is a cognitive enhancer that acts by increasing vigilance and improving learning and memory. We previously reported that nicotine withdrawal up-regulated transcription of some immediately early genes (IEGs), apoptosis-related genes, and signal transduction-related genes in cultures of pheochromocytoma PC12 cells, which are of neuronal lineage (Ichino et al., 1999; Ichino et al., 2002; Yamada et al., 2005). Especially, nicotine withdrawal increased the levels of Bag-1, Rab2, Hsp70, and Raf1 proteins in the cerebral cortex, hippocampus, and striatum in vivo in mice. Egr-1 and Nur77 protein levels were dramatically increased by nicotine withdrawal. (Yamada et al., 2006). Also, there is a study that analyzed the expression of mRNAs by in situ hybridization using a rat genome array after acute intermittent nicotine treatment and found that some mRNA levels of them (Nr4a1, Egr-1 and Egr-2) are significantly up-regulated in the rat cerebral cortex and hippocampal regions (Belluardo et al., 2005). In the present study, we aimed at further elucidating the effects of nicotine withdrawal on the expression of the upstream proteins of IEGs in the brain of mice after chronic administration of nicotine. We observed significant increases in the protein levels of Egr-1 and Nur77 in the specific regions and in specific cells in the mouse brain by Western blot analysis and by immunohistochemistry. The present results suggest that such changes may also occur in the brain of smokers during abstaining from smoking and may be related to some of their symptoms of nicotine withdrawal. © Society of Integrated Sciences.

Catecholamines [dopamine, noradrenaline (norepinephrine), and adrenaline (epinephrine); CAs] are neurotransmitters in the central and peripheral nervous systems as well as hormones in the endocrine system. CAs in the brain play a central role in versatile functions as slow-acting neurotransmitters functioning in synaptic neurotransmission, modulating the effects of fast-acting neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA). In this review, I focus on recent advances in the biochemistry and molecular biology of the CA system in humans in health and disease, especially in neuropsychiatric diseases such as Parkinson's disease (PD), in relation to the biosynthesis of CAs regulated by a pteridine-dependent monooxygenase, tyrosine 3-monooxygenase (tyrosine hydroxylase, TH) and its pteridine cofactor, tetrahydrobiopterin (BH4).

Catecholamines [dopamine, noradrenaline (norepinephrine), and adrenaline (epinephrine); CAs] are neurotransmitters in the central and peripheral nervous systems as well as hormones in the endocrine system. CAs in the brain play a central role in versatile functions as slow-acting neurotransmitters functioning in synaptic neurotransmission, modulating the effects of fast-acting neurotransmitters such as glutamate and gamma-aminobutyric acid (GABA). In this review, I focus on recent advances in the biochemistry and molecular biology of the CA system in humans in health and disease, especially in neuropsychiatric diseases such as Parkinson's disease (PD), in relation to the biosynthesis of CAs regulated by a pteridine-dependent monooxygenase, tyrosine 3-monooxygenase (tyrosine hydroxylase, TH) and its pteridine cofactor, tetrahydrobiopterin (BH4).

Monoamine oxidases A and B (MAO A and MAO B) are the major enzymes that catalyze the oxidative deamination of monoamine neurotaransmitters such as dopamine (DA), noradrenaline, and serotonin in the central and peripheral nervous systems. MAO B is mainly localized in glial cells. MAO B also oxidizes the xenobiotic 1-methyl-4-phenyl-1.2.3,6-tetrahydropyridine (MPP+) to a parkinsonism-producing neurotoxin, 1-methyl-4-phenyl-pyridinium (MPP+). MAO B may be closely related to the pathogenesis of Parkinson's disease (PD), in which neuromelanin-containing DA neurons in the substantia nigra projecting to the striatum in the brain selectively degenerate. MAO B degrades the neurotransmitter DA that is deficient in the nigro-striatal region in PD, and forms H,02 and toxic aldehyde metabolites of DA. H2O2 produces highly toxic reactive oxygen species (ROS) by Fenton reaction that is catalyzed by iron and neuromelanin. MAO B inhibitors such as L-(-)-deprenyl (selegiline) and rasagiline are effective for the treatment of PD. Concerning the mechanism of the clinical efficacy of MAO B inhibitors in PD, the inhibition of DA degradation (a symptomatic effect) and also the prevention of the formation of neurotoxic DA metabolites, i.e., ROS and dopamine derived aldehydes have been speculated. As;mother mechanism of clinical efficacy, MAO B inhibitors such as selegiline are speculated to have neuroprotective effects to prevent progress of PD. The possible mechanism of neuroprotection of MAO B inhibitors may be related not only to MAO B inhibition but also to induction and activation of multiple factors for anti-oxidative stress and anti-apoptosis: i.e., catalase, superoxide dismutase 1 and 2, thioredoxin, Bcl-2, the cellular poly(ADP-ribosyl)ation, and binding to glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Furthermore, it should be noted that selegiline increases production of neurotrophins such as nerve growth factor (NGF), brain-derivec neurotrophic factor (BDNF), and glial cell line-derived neurotrphic factor (GDNF), possibly from glial cells, to protect neurons from inflammatory process.

Parkinson's disease (PD) is a movement disorder caused by degeneration of the nigrostriatal dopamine (DA) neurons in the substantia nigra pars compacta and the resultant deficiency in the neurotransmitter DA at the nerve terminals in the striatum. We and other investigators found increased levels of pro-inflammatory cytokines Such as tumor necrosis factor (TNF)-alpha, interleukin (IL)1lbeta, and IL-6, and decreased levels of neurotrophins such as brain-derived neurotrophic factor (BDNF) in the nigrostriatal region of postmortem brains and/or in the ventricular or lumbar cerebrospinal fluid (CSF) from patients with sporadic PD, and in animal models, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine (6-OHDA)-induced PD. These changes in cytokine and neurotrophin levels may be initiated by activated microglia, which may then promote apoptotic cell death and subsequent phagocytosis of DA neurons. Cytokines as pleiotropic factors, promote signals that either lead to cell death or exert neuroprotective effects. The discovery of toxic changes in trophic microglia by M. Sawada and co-workers is important to this point. Ultimately, microglial cells may regulate Cellular changes that cause either harm or benefit by producing cytokines or neurotrophins depending upon the primary cause and the circumstances during the inflammatory process of PD.

Activation of microglia has been implicated in various neurodegenerative disorders, and thus the inhibition of microglial activity may suppress these disorders. Earlier we designed and synthesized an NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) that showed anti-inflammatory and anti-tumor activities in vivo. In the present research, we studied whether DHMEQ would inhibit the activation of mouse microglial cells. DHMEQ inhibited lipopolysaccharide (LPS)-induced activation of NF-kappaB in an electrophoresis mobility shift assay. It also inhibited LPS-induced secretions of TNF-alpha and IL-6 from mouse microglial cell line 6-1 cells.

We report the results of a collaborative study on malignant syndrome (MS) that developed in patients being treated with levodopa and other anti-parkinsonian drugs. We analyzed clinical features, laboratory findings, precipitating events, and risk factors for poor outcome. The study was conducted in five centers in Japan. Patients who developed MS between January 1991 and December 1997 were included. The enrollment criteria used were the same as those for neuroleptic MS proposed by Levenson et al. (1985). A total of 99 episodes were encountered in 93 patients (72 with Parkinson's disease and 21 with secondary parkinsonism); one patient had four recurrences of MS and three patients had two recurrences. High fever was the most frequent clinical manifestation of MS followed by worsening of parkinsonism, and then altered levels of consciousness. Serum creatine kinase was abnormally elevated in all the patients studied. Life-threatening complications were rhabdomyolysis, disseminated intravascular coagulation, and acute renal failure. The most frequent precipitating event was discontinuation or dose reduction of anti-parkinsoian drugs, particularly levodopa. No drug was the exception in the precipitation of MS. Intercurrent infection was the next most common precipitating event. MS developed without drug withdrawal or infection in some patients. In five patients, severe 'wearing off' phenomenon was the only event preceding the onset of MS. Hot weather and dehydration appeared to be the cause in three patients. Among the total of 99 episodes, patients recovered to the pre-MS state following 68 episodes (68.7%); in the remaining 31.3%, patients failed to recover to their previous state. Older age, higher Hoehn and Yahr stage during the symptomatic phase of MS, higher akinesia score, and the absence of wearing off phenomenon prior to developing MS were associated with poor outcome. The most frequently used treatments of MS were intravenous fluid, levodopa, dantrolene sodium, and intragastric bromocriptine. Early introduction of treatment is important. Any elevation of body temperature during the course of anti-parkinsonian drug treatment should be considered as MS until proved otherwise. (C) 2003 Elsevier Science Ltd. All rights reserved.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine (DA) biosynthesis.(1,2) Exon 3 of the human TH gene encodes the sequence from Ser31 to Glu104 of type 1 enzyme,(3,4) which contains the critical parts for regulation of the catalytic activity. The amino acid residues Gly36-Arg37-Arg38 were identified as a key sequence for DA to exert its inhibitory effect on catalytic activity.(5-7) Therefore, we screened the nucleotide sequences of exon 3 from 201 Japanese patients with schizophrenia to explain the elevation in the synaptic or presynaptic DA concentrations in the schizophrenic brain,(8-11) based on the hypothesis that any mutation changing the amino acid sequence Gly36-Arg37-Arg38 would result in the elevation of DA synthesis, due to a reduced inhibitory effect of DA on the catalytic activity.(5-7) However, no mutated sequences of exon 3 and both exon-intron boundaries were detected in any of the patients examined. Polymorphisms generating Val81 and Met81 were compared of the distributions of genotype and allele between the patients and 175 Japanese healthy controls, which did not suggest an association between the polymorphism and schizophrenia. These results indicate that exon 3 of the human TH gene lacks association with schizophrenia in Japanese patients.

Glial cell line-derived neurotrophic factor (GDNF) was measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by highly sensitive sandwich enzyme-linked immunosorbent assay. In both groups, the levels of GDNF in the various brain regions were lower (pg/mg protein) than those of brain-derived growth factor (ng/mg order), and were significantly higher in the nigro-striatal dopaminergic regions (substantia nigra, caudate nucleus, putamen) than in the cerebellum and frontal cortex (P < 0.05). However, the content of GDNF in the dopaminergic regions showed no significant difference between parkinsonian and control patients. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Dopamine-beta -hydroxylase (D betaH) catalyzes the conversion of dopamine to norepinephrine and is released from sympathetic neurons into the circulation. Plasma-D betaH activity varies widely between individuals, and a subgroup of the population has very low activity levels. Mounting evidence suggests that the DBH structural gene is itself the major quantitative-trait locus (QTL) for plasma-D betaH activity, and a single unidentified polymorphism may account for a majority of the variation in activity levels. Through use of both sequencing-based mutational analysis of extreme phenotypes and genotype/phenotype correlations in samples from African American, European American (EA), and Japanese populations, we have identified a novel polymorphism (-1021C-->T), in the 5' flanking region of the DBH gene, that accounts for 35%-52% of the variation in plasma-DbH activity in these populations. In EAs, homozygosity at the T allele predicted the very low D betaH-activity trait, and activity values in heterozygotes formed an intermediate distribution, indicating codominant inheritance. Our findings demonstrate that -1021C-->T is a major genetic marker for plasma-D betaH activity and provide new tools for investigation of the role of both DbH and the DBH gene in human disease.

The structure of the native pteridine in Tetrahymena pyriformis was determined as (6R)-5,6,7,8-tetrahydro-D-monapterin (=(6R)-2-amino-5.6.7.8-tetrahydro-6-[(1R,2R)-1,2,3-trihydroxypropyl)]pteridin-4(3II)-one:4). First, the configuration of the 1.2.3-trihydroxypropyl side chain was confirmed as D-threo by the fluorescence-detected circular dichroism (FDCD) spectrum of its aromatic pterin derivative 2 obtained by 1(2) oxidation (fig. 1.). The configuration at the 6-position of 4 was determined as (R) by comparison of its hexaacetyl derivative 6 with authentic (6R)- and (6S)-hexaacetyl-5,6,7,8-tetrahydro-D-monapterins 6 and 7, respectvely, in the HPLC, LC/MS and LC-MS.MS (figs. 3-6), (6R)-5,6,7,8-Tetrahydro-D-monapterin (4) is a newly discovered natural tetrahydropterin.

The major pterin from Escherichia coli was determined as L-monapterin, by applying fluorescence detected circular dichroism (FDCD). FDCD was highly sensitive and specific to fluorescent chiral pterin, and allowed structure determination even in the presence of non-fluorescent contaminants.